Assessment of the Cytotoxic Effect of a Series of 1,4-Dihydropyridine Derivatives Against Human Cancer Cells

Cancer is a leading cause of death worldwide. Despite the availability of several chemotherapeutic drugs, there is still a great need for more efficient agents for a better management of cancer. In this contribution, a series of 11 1,4-dihydropyridines (1,4-DHPs) (4a, 4b and 7a-i) were synthesized and evaluated for their cytotoxic effect against MCF-7, LS180 and MOLT-4 cancer cell lines using MTT assay. Synthesized 2,6-dimethyl-3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-dihydropyridines exhibited different potencies ranging from weak to good cytotoxic activities, while no activity could be recorded for 1,4-bis(2,6-dimethyl-3,5-dialkyloxylcarbonyl,4-dihydropyridine-4-yl) benzene compounds (4a and 4b). Tested DHP derivatives were more potent against MOLT-4 cells, when compared to LS180 and MCF-7 cells. Compounds 7d (IC50 = 28.5 ± 3.5 µM), 7a (IC50 = 29.7 ± 4.7 µM) and 7a (IC50 = 17.4 ± 2.0 µM) were the most potent derivatives against MCF-7, LS180 and MOLT-4 cells, respectively. It appeared that the introduction of N-thiazolyl carbamoyl group at the C3 and C5 positions of DHP ring enhanced the cytotoxic potential of these derivatives (compounds 7a-e). The findings of this study suggest that some of the thiazole substituted 1,4-DHPs may be candidates for further modifications towards the discovery of potent anticancer agents.


Introduction
Cancer is characterized by uncontrolled cell growth and division. Nowadays cancer is an important cause of mortality worldwide that affects a huge number of people during their life (1). On the basis of recent reports, approximately 17 million cancer deaths per year might occur by 2030 (2). Despite the remarkable progress in the prevention and treatment of cancer, chemotherapy methods are hindered with significant limitations (3). Therefore development of efficient and selective antitumor agents is one of the fundamental challenges of researchers working in the field of pharmaceutical sciences.

Materials and methods Chemistry
All the synthesized compounds were characterized by mass spectroscopy, IR and 1 H NMR. IR spectra were recorded on a Nicolet FT-IR Magna 550 spectrophotometer. 1 H NMR spectra were determined by a Bruker FT-500 MHz spectrometer in chloroform-d 1 or DMSO-d 6 . All the chemical shifts were reported as δ values (ppm) against tetramethylsilane as an internal standard. The MS spectra were recorded using a Finnigan TSQ-70 spectrometer at 70 eV. CHN/CHNS analysis was performed using CHNS-932 Leco analyzer and the results were within ± 0.4% of the theoretical values.
The progress of the reaction was monitored by TLC. On completion of the reaction, the solvent was removed to some extent under reduced pressure. The product was filtered, washed with small portions of cold methanol and then dried to afford the final product 4b. As for compound 4a, similar procedure was carried out while further purification by column Scheme 1. Chemical structure of dexniguldipine. chromatography and preparative TLC using ethylacetate/petroleum ether as eluent afforded the pure product. Physical characteristics data of the final compounds are summarized in Table 1.
Cell lines MCF-7 (human breast adenocarcinoma), LS180 (human colon adenocarcinoma), and MOLT-4 (human acute lymphoblastic leukemia) cells were obtained from the National Cell Bank of Iran, Pasteur Institute, Tehran, Iran. All cell lines were maintained in RPMI 1640 supplemented with 10% FBS, and 100 units/ mL penicillin-G and 100 m g/mL streptomycin. Cells were grown in monolayer (MCF-7 and LS180) or in suspension (MOLT-4) cultures.

Cytotoxic effect
Cell viability following exposure to synthetic compounds was estimated by using the MTT reduction assay (22, 27). LS180 and MOLT-4 cells were plated in 96-well microplates at a density of 5 × 10 4 cells/mL (100 μL per well), while MCF-7 cells were plated at a density of 3 × 10 4 cells/mL. Control wells contained no drugs and blank wells contained only growth medium for background correction. After overnight incubation at 37 °C, half of the growth medium was removed and 50 μL of medium supplemented with 10, 25, 50 and 100 μM of synthetic compounds were added. Compounds were all first dissolved in DMSO and then diluted in medium so that the maximum concentration of DMSO in the wells did not exceed 0.5%. Cells were further incubated for 72 h. At the end of the incubation time, the medium was removed and MTT was added to each well at a final concentration of 0.5 mg/mL and plates were incubated for another 4 h at 37 °C. Then formazan crystals were solubilized in 200 μL DMSO. The optical density was measured at 570 nm with background correction at 655 nm using Table 2. Chemical structures of the 2,6-dimethyl-3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-dihydropyridine derivatives (VIIa-i).
Considering the data in Table 3, the following structure activity relationships (SAR) may be envisaged: DHP compounds bearing 3,5-bis-N-(4methyl-2-thiazolyl) substituents (7a-e) showed superior cytotoxic effect compared to 3,5-bis-N-(5-methyl-3-isoxazolyl) substituted DHPs (7fg). Higher activity of thiazole containing DHPs might be attributed to the role of sulfur atoms in enhancing the lipophilicity of the molecule and thus enhancing the penetration through cell membranes.
3,5-bis-N-(6-Ethoxy-2-benzothiazolyl) substituted DHP molecule (7i) did not show cytotoxic activity against MCF-7, LS180 and MOLT-4 cancer cell lines. This activity loss might be attributed to the possible steric hindrance of this derivative at the target site.
1 , 4 -b i s ( 2 , 6 -d i m e t h y l -3 , 5dialkyloxylcarbonyl-1,4-dihydropyridine-4-yl) benzenes (4a and 4b) had no cytotoxic activity against any of the tested cells. One possible explanation to this is their bulky chemical structure leading to steric clash in their site of action.
Similar trend of activity could be observed for derivatives 7a-i against the LS180 and MOLT-4 cells. Due to the structural similarities of these compounds, observed order of activity could be related to their different substitution pattern on C4 of DHP ring (4-nitrophenyl > 4-methoxyphenyl > 2-furyl > 6-methyl-2pyridil > 2-nitrophenyl). It appeared that DHP derivatives possessing H-acceptor/donor groups on their para position of C4-substituted aromatic ring showed superior activity against LS180 and MOLT-4 cells (Table 3.).

Conclusion
2,6-Dimethyl-3,5-bis-N-(aryl/heteroaryl) c a r b a m o y l -4 -a r y l -1 , 4 -d i h y d r o p y r i d i n e analogues showed weak to relatively good cytotoxic activity against MCF-7, LS180 and MOLT-4 human cancer lines, while a superior potency was observed in the case of MOLT-4 cells (7a: IC 50 = 17.4±2.0 µM). It was revealed that 1,4-bis(2,6-dimethyl-3,5dialkyloxylcarbonyl-1,4 dihydropyridine-4-yl) benzenes (4a and 4b) exhibited no cytotoxic effect on tested cancer cell lines possibly due to their bulky scaffold and hence steric hindrance in their site of action. The outcomes of this study may provide helpful information to guide the rational design and synthesis of more potent cytotoxic molecules on the basis of 1,4-DHP scaffold.